The present invention relates to the microbiological industry. In particular, the present invention relates to the development of a new approach to regulated gene expression in bacterial cells.
Induction of expression of genes cloned into recombinant bacteria (e.g., E. coli) by the addition of the relatively simple and cheap chemical compounds is a very attractive idea for many biotechnological processes. Among the group of potential attractive inducer candidates are natural L-amino acids.
To the knowledge of the present inventors, the regulatory regions of the tryptophanase genes represent unique natural systems induced by addition of tryptophan to a cultural medium [Landick R., Turnbough C. L., Yanofsky C. “Transcription attenuation”/ In: “Escherichia coli and Salmonella. Cellular and molecular biology” (Second Edition, F. C. Neidhardt—Editor in Chief), (1996), pp. 1263-1286]. In contrast, there are the several known systems in which the controlled gene expression is decreased in the presence of an excess of amino acids in the cells (for example, the system of the trp-operon repressor-operator [Platt, T. “Regulation of gene expression in the tryptophan operon of Escherichia coli”/In: “The Operon” (Miller, J. H., Reznikoff, W. S.—Eds.), Cold Spring Harbor Laboratory (1978), pp. 263-302.], and the attenuation of amino acid operon transcription [Landick R., Turnbough C. L., Yanofsky C. “Transcription attenuation”/ In: “Escherichia coli and Salmonella. Cellular and molecular biology” (Second Edition, F. C. Neidhardt—Editor in Chief), (1996), pp. 1263-1286]).
The molecular mechanism of transcription attenuation in amino acid operons is based on the possibility of alternative mRNA secondary structure formation in the “attenuator” region as a result of translation of the “leader” peptide located upstream from the first structural gene of the operon. The coding region of the leader peptide gene is enriched by the codons of the sense amino acid (i.e., the codon of those amino acid whose biosynthesis is provided by the corresponding gene product encoded by the operon). For example, for the trp-operon, the leader peptide gene is enriched with Trp-codons, for the his-operon—His-codons, for the thr-operon—Thr-codons and Ile-codons, etc).
The details of this well-established regulation of transcription are presented in the FIG. 1, using the attenuator regions of the E. coli trp- and his-operons as examples. As shown in FIG. 1, alternative mRNA secondary structures can be formed during transcription of the corresponding DNA fragments: the hairpins t1:t2 and t3:t4, or their alternative—t2:t3, can be formed for trp-leader, analogously, h1:h2, h3:h4 and h5:h6, or h2:h3 and h4:h5 can be formed for his-leader. The hairpins t3:t4 and h5:h6 are the typical ρ-independent transcription terminators. Accordingly, their formation during the elongation of transcription leads to termination in the attenuator regions of the corresponding operon preventing the expression of the structural genes of the operon.
The process of mRNA secondary structure formation is coupled to the process of mRNA synthesis. Therefore, the hairpins t1:t2 and then t3:t4 are formed step by step (its alternative—the hairpin t2:t3 could not be formed because t1:t2 has already been formed) in the trp-attenuator when the leader peptide has not been translated. Analogously, rapid formation of, in succession, the hairpins h1:h2, h3:h4, and h5:h6 prevents the formation of their alternative structures—h2:h3 and h4:h5 during synthesis of his-attenuator mRNA without translation of the corresponding leader peptide.
As discussed above, the formation of the hairpins t3:t4, as well as h5:h6 leads to termination in the corresponding attenuator regions. This situation could be realized during in vitro transcription of the corresponding DNAs by a pure RNA polymerase without any translational factors in the reaction mixture, or in vivo under general amino acid starvation conditions.
A much more complicate situation may occur in vivo during translation of the leader peptide when excessive sense amino acid is present (more precisely, the corresponding charged tRNA is in excess), or a deficiency of the same occurs in the bacterial cell. It has been shown that RNA polymerase initiating attenuator mRNA transcription stops at the pause site in the region located immediately downstream from the hairpin 1:2 (probably, the formation of this hairpin is the essential, but not the adequate condition of such pausing) [Chan, C. L., Wang, D., Landick, R. “Multiple interactions stabilize a single paused transcription intermediate in which hairpin to 3′ end spacing distinguishes pause and termination pathway”/ J. Mol. Biol. 268 (1997) 54-68]. The ribosome translating the N-terminal part of the leader peptide releases the RNA polymerase followed by continuation of transcriptional elongation and, thus, enabling the formation of the alternative mRNA hairpin (2:3). The following events depend on the intracellular concentration of the charged-tRNA(s) of the sense amino acid, because the corresponding codons of the leader peptide gene have to be translated by the ribosome following charging of the respective tRNA.
Under sense amino acid starvation conditions, the ribosome stalls at the sense codon and the hairpin 2:3 is left uninterrupted. In the scenario, while RNA polymerase synthesizes the downstream fragment of the mRNA which, in principle, could form the terminator hairpin (t3:t4—for the trp-attenuator and h5:h6—for the his-attenuator), but it does not fold due to the existence of the alternative hairpin (t2:t3—for the trp, and the structure h2:h3, h4:h5—for the his), followed by transcription elongation and synthesis of the mRNA of the operon structural genes.
In the presence of an excess of the sense amino acid, the translation of the leader peptide occurs with high efficiency. In this scenario, the ribosome initially disrupting the hairpin 1:2, disrupts the hairpin 2:3, as well, and stalls at the stop codon of the leader peptide. The stall at the stop codon of the leader peptide leads to formation of the terminator hairpin and to the termination of transcription in the attenuator region.